WO2012056401A1 - Polynucléotides isolés exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgéniques les comprenant et leurs utilisations pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante. - Google Patents

Polynucléotides isolés exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgéniques les comprenant et leurs utilisations pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante. Download PDF

Info

Publication number
WO2012056401A1
WO2012056401A1 PCT/IB2011/054763 IB2011054763W WO2012056401A1 WO 2012056401 A1 WO2012056401 A1 WO 2012056401A1 IB 2011054763 W IB2011054763 W IB 2011054763W WO 2012056401 A1 WO2012056401 A1 WO 2012056401A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
nucleic acid
seq
polynucleotide
use efficiency
Prior art date
Application number
PCT/IB2011/054763
Other languages
English (en)
Inventor
Rudy Maor
Iris Nesher
Original Assignee
Rosetta Green Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rosetta Green Ltd. filed Critical Rosetta Green Ltd.
Priority to US13/881,437 priority Critical patent/US20140013469A1/en
Priority to CA2815769A priority patent/CA2815769A1/fr
Priority to AU2011322146A priority patent/AU2011322146A1/en
Priority to EP11799820.3A priority patent/EP2633056A1/fr
Publication of WO2012056401A1 publication Critical patent/WO2012056401A1/fr
Priority to IL225964A priority patent/IL225964A0/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention in some embodiments thereof, relates to isolated polynucleotides expressing or modulating microRNAs or targets of same, transgenic plants comprising same and uses thereof in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.
  • Plant growth is reliant on a number of basic factors: light, air, water, nutrients, and physical support. All these factors, with the exception of light, are controlled by soil to some extent, which integrates non-living substances (minerals, organic matter, gases and liquids) and living organisms (bacteria, fungi, insects, worms, etc.). The soil's volume is almost equally divided between solids and water/gases.
  • An adequate nutrition in the form of natural as well as synthetic fertilizers may affect crop yield and quality, and its response to stress factors such as disease and adverse weather. The great importance of fertilizers can best be appreciated when considering the direct increase in crop yields over the last 40 years, and the fact that they account for most of the overhead expense in agriculture.
  • Sixteen natural nutrients are essential for plant growth, three of which, carbon, hydrogen and oxygen, are retrieved from air and water. The soil provides the remaining 13 nutrients.
  • Nutrients are naturally recycled within a self-sufficient environment, such as a rainforest. However, when grown in a commercial situation, plants consume nutrients for their growth and these nutrients need to be replenished in the system. Several nutrients are consumed by plants in large quantities and are referred to as macronutrients. Three macronutrients are considered the basic building blocks of plant growth, and are provided as main fertilizers; Nitrogen (N), Phosphate (P) and Potassium (K). Yet, only nitrogen needs to be replenished every year since plants only absorb approximately half of the nitrogen fertilizer applied. A proper balance of nutrients is crucial; when too much of an essential nutrient is available, it may become toxic to plant growth. Utilization efficiencies of macronutrients directly correlate with yield and general plant tolerance, and increasing them will benefit the plants themselves and the environment by decreasing seepage to ground water.
  • Nitrogen is responsible for biosynthesis of amino and nucleic acids, prosthetic groups, plant hormones, plant chemical defenses, etc, and thus is utterly essential for the plant. For this reason, plants store nitrogen throughout their developmental stages, in the specific case of corn during the period of grain germination, mostly in the leaves and stalk.
  • NUE nitrogen use efficiency
  • nitrogen supply needs to be replenished at least twice during the growing season. This requirement for fertilizer refill may become the rate-limiting element in plant growth and increase fertilizer expenses for the farmer. Limited land resources combined with rapid population growth will inevitably lead to added increase in fertilizer use.
  • the major agricultural crops (corn, rice, wheat, canola and soybean) account for over half of total human caloric intake, giving their yield and quality vast importance. They can be consumed either directly (eating their seeds which are also used as a source of sugars, oils and metabolites), or indirectly (eating meat products raised on processed seeds or forage).
  • Various factors may influence a crop's yield, including but not limited to, quantity and size of the plant organs, plant architecture , vigor (e.g. seedling), growth rate, root development, utilization of water and nutrients (e.g., nitrogen), and stress tolerance.
  • Plant yield may be amplified through multiple approaches; (1) enhancement of innate traits (e.g., dry matter accumulation rate, cellulose/lignin composition), (2) improvement of structural features (e.g., stalk strength, meristem size, plant branching pattern), and (3) amplification of seed yield and quality (e.g., fertilization efficiency, seed development, seed filling or content of oil, starch or protein).
  • enhancement of innate traits e.g., dry matter accumulation rate, cellulose/lignin composition
  • structural features e.g., stalk strength, meristem size, plant branching pattern
  • amplification of seed yield and quality e.g., fertilization efficiency, seed development, seed filling or content of oil, starch or protein.
  • Root morphogenesis has already shown to increase tolerance to low phosphorus availability in soybean (Miller et al, (2003), Funct Plant Biol 30:973-985) and maize (Zhu and Lynch (2004), Funct Plant Biol 31 :949-958).
  • genes governing enhancement of root architecture may be used to improve NUE and drought tolerance.
  • An example for a gene associated with root developmental changes is ANR1, a putative transcription factor with a role in nitrate (N03 ) signaling.
  • ANR1 a putative transcription factor with a role in nitrate (N03 ) signaling.
  • Abiotic stress refers to a range of suboptimal conditions as water deficit or drought, extreme temperatures and salt levels, and high or low light levels. High or low nutrient level also falls into the category of abiotic stress.
  • the response to any stress may involve both stress specific and common stress pathways (Pastori and Foyer (2002), Plant Physiol, 129: 460-468), and drains energy from the plant, eventually resulting in lowered yield.
  • stress specific and common stress pathways Pieris (2002), Plant Physiol, 129: 460-468
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 21, 22, 23-37, 38-52, 1209, 1211, 1212, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • a transgenic plant exogenously expressing a polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 23-37, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant.
  • said exogenous polynucleotide encodes a precursor of said nucleic acid sequence.
  • said precursor of said nucleic acid sequence is at least 60 % identical to SEQ ID NO: 21, 22, 38-52, 1209, 1211, 1212.
  • said exogenous polynucleotide encodes a miRNA or a precursor thereof.
  • said exogenous polynucleotide encodes a siRNA or a precursor thereof.
  • said exogenous polynucleotide is selected from the group consisting of SEQ ID NO: 10, 6-9, 21, 22, 23- 37, 38-52, 1209, 1211, 1212.
  • an isolated polynucleotide having a nucleic acid sequence at least 90 % identical to SEQ ID NO: 6, 7 and 9, wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of a plant.
  • said nucleic acid sequence is selected from the group consisting of SEQ ID NO: 6, 7 and 9.
  • said polynucleotide encodes a precursor of said nucleic acid sequence.
  • said polynucleotide encodes a miRNA or a precursor thereof.
  • said polynucleotide encodes a siRNA or a precursor thereof.
  • nucleic acid construct comprising the isolated polynucleotide above under the regulation of a cis-acting regulatory element.
  • said cis-acting regulatory element comprises a promoter
  • said promoter comprises a tissue-specific promoter.
  • said tissue-specific promoter comprises a root specific promoter.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.
  • a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209.
  • an isolated polynucleotide which downregulates an activity or expression of a gene encoding an RNAi molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209.
  • said polynucleotide encodes a miRNA-Resistant Target as set forth in SEQ ID NO 1104-1124.
  • said isolated polynucleotide encodes a target mimic as set forth in SEQ ID NO: 18 or 19.
  • nucleic acid construct comprising the isolated polynucleotide above under the regulation of a cis-acting regulatory element.
  • said cis-acting regulatory element comprises a promoter
  • said promoter comprises a tissue-specific promoter.
  • said tissue-specific promoter comprises a root specific promoter.
  • the method further comprises growing the plant under limiting nitrogen conditions.
  • the method further comprises growing the plant under abiotic stress.
  • said abiotic stress is selected from the group consisting of salinity, drought, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.
  • the plant is a monocotyledon.
  • the plant is a dicotyledon.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • a transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.
  • nucleic acid construct comprising a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 927-1021, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, and wherein said polynucleotide is under a transcriptional control of a cis-acting regulatory element.
  • said polynucleotide is selected from the group consisting of SEQ ID NO: 1022-1090.
  • said polypeptide is selected from the group consisting of SEQ ID NO: 927-1021.
  • said cis-acting regulatory element comprises a promoter
  • said promoter comprises a tissue-specific promoter.
  • said tissue-specific promoter comprises a root specific promoter.
  • the method further comprises growing the plant under limiting nitrogen conditions.
  • the method further comprises growing the plant under abiotic stress.
  • said abiotic stress is selected from the group consisting of salinity, drought, water deprivation, flood, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, atmospheric pollution and UV irradiation.
  • the plant is a monocotyledon.
  • the plant is a dicotyledon.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • a transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.
  • nucleic acid construct comprising a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 % homologous to SEQ ID NOs: 854-894, wherein said polypeptide is capable of regulating nitrogen use efficiency of a plant, said nucleic acid sequence being under the regulation of a cis-acting regulatory element.
  • said polynucleotide acts by a mechanism selected from the group consisting of sense suppression, antisense suppresion, ribozyme inhibition, gene disruption.
  • said cis-acting regulatory element comprises a promoter
  • said promoter comprises a tissue-specific promoter.
  • said tissue-specific promoter comprises a root specific promoter.
  • all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.
  • methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control.
  • the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
  • FIG. 1 is a scheme of a binary vector that can be used according to some embodiments of the invention.
  • FIGs. 2 A- J are schematic illustrations of some of the miRNA sequences which may be used in accordance with the present invention.
  • the present invention in some embodiments thereof, relates to isolated polynucleotides expressing or modulating microRNAs or targets of same, transgenic plants comprising same and uses thereof in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant.
  • N fertilizers The doubling of agricultural food production worldwide over the past four decades has been associated with a 7-fold increase in the use of nitrogen (N) fertilizers.
  • N nitrogen
  • the most typical examples of such an impact are the eutrophication of freshwater and marine ecosystems as a result of leaching when high rates of nitrogen fertilizers are applied to agricultural fields.
  • NUE plant nitrogen use efficiency
  • microRNA miR A sequences that are differentially expressed in maize plants grown under nitrogen limiting conditions versus maize plants grown under conditions wherein nitrogen is a non-limiting factor.
  • miRNA miRNA sequences that are upregulated or downregulated in roots and leaves, and suggest using same or sequences controlling same in the generation of transgenic plants having improved nitrogen use efficiency.
  • the present inventors have analyzed the level of expression of the identified miRNA sequences under optima, and nitrogen deficient conditions by quantitiative RT-PCR and validated the correlation between miRNA expression nitrogen availability.
  • the newly uncovered miRNA sequences relay their effect by affecting at least one of:
  • root architecture so as to increase nutrient uptake; activation of plant metabolic pathways so as to maximize nitrogen absorption or localization; or alternatively or additionally
  • Each of the above mechanisms may affect water uptake as well as salt absorption and therefore embodiments of the invention further relate to enhancement of abiotic stress tolerance, biomass, vigor or yield of the plant.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide having a nucleic acid sequence at least 80 %, 85 %, 90 % or 95 % identical to SEQ ID NOs: 10, 6-9 and 23-37 wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • the exogenous polynucleotide has a nucleic acid sequence at least 90 % identical to SEQ ID NOs: 10, 6-9, 23-37.
  • the exogenous polynucleotide has a nucleic acid sequence at least 95 % identical to SEQ ID NOs: 10, 6-9, 23-37.
  • the exogenous polynucleotide has a nucleic acid sequence as set forth in SEQ ID NOs: 10, 6-9, 23-37.
  • NUE nitrogen use efficiency
  • FUE Fertilizer use efficiency
  • Crop production can be measured by biomass, vigor or yield.
  • the plant's nitrogen use efficiency is typically a result of an alteration in at least one of the uptake, spread, absorbance, accumulation, relocation (within the plant) and use of nitrogen absorbed by the plant.
  • Improved NUE is with respect to that of a non- transgenic plant (i.e., lacking the transgene of the transgenic plant) of the same species and of the same developmental stage and grown under the same conditions.
  • nitrogen-limiting conditions refers to growth conditions which include a level (e.g., concentration) of nitrogen (e.g., ammonium or nitrate) applied which is below the level needed for optimal plant metabolism, growth, reproduction and/or viability.
  • abiotic stress refers to any adverse effect on metabolism, growth, viability and/or reproduction of a plant.
  • Abiotic stress can be induced by any of suboptimal environmental growth conditions such as, for example, water deficit or drought, flooding, freezing, low or high temperature, strong winds, heavy metal toxicity, anaerobiosis, high or low nutrient levels (e.g. nutrient deficiency), high or low salt levels (e.g.
  • Abiotic stress may be a short term effect (e.g. acute effect, e.g. lasting for about a week) or alternatively may be persistent (e.g. chronic effect, e.g. lasting for example 10 days or more).
  • the present invention contemplates situations in which there is a single abiotic stress condition or alternatively situations in which two or more abiotic stresses occur.
  • the abiotic stress refers to salinity
  • the abiotic stress refers to drought.
  • abiotic stress tolerance refers to the ability of a plant to endure an abiotic stress without exhibiting substantial physiological or physical damage (e.g. alteration in metabolism, growth, viability and/or reproductivity of the plant).
  • biomass refers to the amount (e.g., measured in grams of air-dry tissue) of a tissue produced from the plant in a growing season.
  • An increase in plant biomass can be in the whole plant or in parts thereof such as aboveground (e.g. harvestable) parts, vegetative biomass, roots and/or seeds.
  • vigor As used herein the term/phrase “vigor”, “vigor of a plant” or “plant vigor” refers to the amount (e.g., measured by weight) of tissue produced by the plant in a given time. Increased vigor could determine or affect the plant yield or the yield per growing time or growing area. In addition, early vigor (e.g. seed and/or seedling) results in improved field stand.
  • yield refers to the amount (e.g., as determined by weight or size) or quantity (e.g., numbers) of tissues or organs produced per plant or per growing season. Increased yield of a plant can affect the economic benefit one can obtain from the plant in a certain growing area and/or growing time. According to an exemplary embodiment the yield is measured by cellulose content.
  • the yield is measured by oil content.
  • the yield is measured by protein content.
  • the yield is measured by seed number per plant or part thereof (e.g., kernel).
  • a plant yield can be affected by various parameters including, but not limited to, plant biomass; plant vigor; plant growth rate; seed yield; seed or grain quantity; seed or grain quality; oil yield; content of oil, starch and/or protein in harvested organs (e.g., seeds or vegetative parts of the plant); number of flowers (e.g. florets) per panicle (e.g. expressed as a ratio of number of filled seeds over number of primary panicles); harvest index; number of plants grown per area; number and size of harvested organs per plant and per area; number of plants per growing area (e.g. density); number of harvested organs in field; total leaf area; carbon assimilation and carbon partitioning (e.g. the distribution/allocation of carbon within the plant); resistance to shade; number of harvestable organs (e.g. seeds), seeds per pod, weight per seed; and modified architecture [such as increase stalk diameter, thickness or improvement of physical properties (e.g. elasticity)] .
  • the term “improving” or “increasing” refers to at least about 2 %, at least about 3 %, at least about 4 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, at least about 90 % or greater increase in NUE, in tolerance to abiotic stress, in yield, in biomass or in vigor of a plant, as compared to a native or wild-type plants [i.e., plants not genetically modified to express the biomolecules (polynucleotides) of the invention, e.g., a non-transformed plant of the same species and of the same developmental stage which is grown under the same growth conditions as the transformed plant].
  • Improved plant NUE is translated in the field into either harvesting similar quantities of yield, while implementing less fertilizers, or increased yields gained by implementing the same levels of fertilizers.
  • improved NUE or FUE has a direct effect on plant yield in the field.
  • plant encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and isolated plant cells, tissues and organs.
  • the plant may be in any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.
  • plant cell refers to plant cells which are derived and isolated from disintegrated plant cell tissue or plant cell cultures.
  • plant cell culture refers to any type of native (naturally occurring) plant cells, plant cell lines and genetically modified plant cells, which are not assembled to form a complete plant, such that at least one biological structure of a plant is not present.
  • the plant cell culture of this aspect of the present invention may comprise a particular type of a plant cell or a plurality of different types of plant cells. It should be noted that optionally plant cultures featuring a particular type of plant cell may be originally derived from a plurality of different types of such plant cells.
  • Plants that are particularly useful in the methods of the invention include all plants which belong to the super family Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna in
  • the plant used by the method of the invention is a crop plant including, but not limited to, cotton, Brassica vegetables, oilseed rape, sesame, olive tree, palm oil, banana, wheat, corn or maize, barley, alfalfa, peanuts, sunflowers, rice, oats, sugarcane, soybean, turf grasses, barley, rye, sorghum, sugar cane, chicory, lettuce, tomato, zucchini, bell pepper, eggplant, cucumber, melon, watermelon, beans, hibiscus, okra, apple, rose, strawberry, chile, garlic, pea, lentil , canola, mums, arabidopsis, broccoli, cabbage, beet, quinoa, spinach, squash, onion, leek, tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, and also plants used in horticulture, floriculture or forestry, such as, but not limited to, poplar,
  • the plant comprises corn.
  • the plant comprises sorghum.
  • exogenous polynucleotide refers to a heterologous nucleic acid sequence which may not be naturally expressed within the plant or which overexpression in the plant is desired.
  • the exogenous polynucleotide may be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA) molecule.
  • RNA ribonucleic acid
  • the exogenous polynucleotide may comprise a nucleic acid sequence which is identical or partially homologous to an endogenous nucleic acid sequence of the plant.
  • the present teachings are based on the identification of miRNA sequences which modulate nitrogen use efficiency of plants.
  • the exogenous polynucleotide encodes a miRNA or a precursor thereof.
  • microRNA also referred to herein interchangeably as “miRNA” or “miR”
  • miRNA miRNA
  • the phrase “microRNA” or “miR” or a precursor thereof refers to a microRNA (miRNA) molecule acting as a post-transcriptional regulator.
  • the miRNA molecules are RNA molecules of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and which direct the cleavage of another RNA molecule, wherein the other RNA molecule comprises a nucleotide sequence essentially complementary to the nucleotide sequence of the miRNA molecule.
  • a miRNA molecule is processed from a "pre-miRNA” or as used herein a precursor of a pre-miRNA molecule by proteins, such as DCL proteins, present in any plant cell and loaded onto a RISC complex where it can guide the cleavage of the target RNA molecules.
  • proteins such as DCL proteins
  • Pre-microRNA molecules are typically processed from pri-microRNA molecules (primary transcripts).
  • the single stranded RNA segments flanking the pre- microRNA are important for processing of the pri-miRNA into the pre-miRNA.
  • the cleavage site appears to be determined by the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).
  • a "pre-miRNA” molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a double stranded RNA stem and a single stranded RNA loop (also referred to as "hairpin") and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem.
  • the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem.
  • the length and sequence of the single stranded loop region are not critical and may vary considerably, e.g.
  • RNA molecules can be predicted by computer algorithms conventional in the art such as mFOLD.
  • the particular strand of the double stranded RNA stem from the pre-miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation.
  • Naturally occurring miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules but they can also be introduced into existing pre- miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest.
  • the scaffold of the pre-miRNA can also be completely synthetic.
  • synthetic miRNA molecules may be comprised within, and processed from, existing pre-miRNA molecule scaffolds or synthetic pre- miRNA scaffolds.
  • pre-miRNA scaffolds may be preferred over others for their efficiency to be correctly processed into the designed microRNAs, particularly when expressed as a chimeric gene wherein other DNA regions, such as untranslated leader sequences or transcription termination and polyadenylation regions are incorporated in the primary transcript in addition to the pre-microRNA.
  • the miRNA molecules may be naturally occurring or synthetic.
  • the present teachings contemplate expressing an exogenous polynucleotide having a nucleic acid sequence at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NOsl-10, 23-37, 57-449, provided that they regulate nitrogen use efficiency.
  • the present teachings contemplate expressing an exogenous polynucleotide having a nucleic acid sequence at least 65%, 50 %, 75 %, 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NOs. 1-10, 21 and 22 (mature and precursors Tables 1 and 3, and Figures 2A-H representing the core maize genes), provided that they regulate nitrogen use efficiency.
  • Tables 1 and 3 below illustrates exemplary miRNA sequences and precursors thereof which over expression are associated with modulation of nitrogen use efficiency.
  • the present invention envisages the use of homologous and orthologous sequences of the above miRNA molecules.
  • use of homologous sequences can be done to a much broader extend.
  • the degree of homology may be lower in all those sequences not including the mature miRNA segment therein.
  • stem-loop precursor refers to stem loop precursor RNA structure from which the miRNA can be processed.
  • Pre-microRNA molecules are typically processed from pri-microRNA molecules (primary transcripts).
  • the single stranded RNA segments flanking the pre- microRNA are important for processing of the pri-miRNA into the pre-miRNA.
  • the cleavage site appears to be determined by the distance from the stem-ssRNA junction (Han et al. 2006, Cell 125, 887-901, 887-901).
  • a "pre -miRNA” molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a double stranded RNA stem and a single stranded RNA loop (also referred to as "hairpin") and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem.
  • the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem.
  • the length and sequence of the single stranded loop region are not critical and may vary considerably, e.g.
  • RNA molecules between 30 and 50 nt in length.
  • the complementarity between the miRNA and its complement need not be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated.
  • the secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD.
  • the particular strand of the double stranded RNA stem from the pre -miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation.
  • the exogenous polynucleotide encodes a stem-loop precursor of the nucleic acid sequence.
  • a stem-loop precursor can be at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, at least about 95 % or more identical to SEQ ID NOs: 21-22, 38-52, 1209, 1211, 1212, 454-846, 53- 56, 1209 (homologs precursor Tables 1 and 3 and Figures 2A-H), provided that it regulates nitrogen use efficiency.
  • Identity e.g., percent identity
  • NCBI National Center of Biotechnology Information
  • Homology e.g., percent homology, identity + similarity
  • NCBI National Center of Biotechnology Information
  • the term “homology” or “homologous” refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences.
  • Homologous sequences include both orthologous and paralogous sequences.
  • paralogous relates to gene-duplications within the genome of a species leading to paralogous genes.
  • orthologous relates to homologous genes in different organisms due to ancestral relationship.
  • One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov.
  • the blast results may be filtered.
  • the full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequences of the organism from which the sequence-of- interest is derived.
  • the results of the first and second blasts are then compared.
  • An orthologue is identified when the sequence resulting in the highest score (best hit) in the first blast identifies in the second blast the query sequence (the original sequence-of- interest) as the best hit. Using the same rational a paralogue (homolog to a gene in the same organism) is found.
  • the ClustalW program may be used [Hypertext Transfer Protocol ://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor-joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.
  • an isolated polynucleotide having a nucleic acid sequence at least 80 %, 85 % or preferably 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 % 99 % or 100 % identical to SEQ ID NO: 6, 7, 9, 1209, 1210, 1211, 1212 (Table 1 predicted both upregulated and downregulated), wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of a plant.
  • the isolated polynucleotide encodes a stem-loop precursor of the nucleic acid sequence.
  • the stem-loop precursor is at least about 60
  • RNAi sequences which are down regulated under nitrogen limiting conditions.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a gene encoding a miRNA molecule having a nucleic acid sequence at least 80 %, 85 % or preferably 90 %, 95 % or even 100 % identical to the sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 53-56, 1209, 57-449, 454-846 (Tables 1 and 4 down-regulated), thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant
  • down-regulation refers to reduced activity or expression of the miRNA (at least 10 %, 20 %, 30 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % reduction in activity or expression) as compared to its activity or expression in a plant of the same species and the same developmental stage not expressing the exogenous polynucleotide.
  • Nucleic acid agents that down-regulate miR activity include, but are not limited to, a target mimic, a micro-RNA resistant gene and a miRNA inhibitor.
  • the target mimic or micro-RNA resistant target is essentially complementary to the microRNA provided that one or more of following mismatches are allowed:
  • the target mimic RNA is essentially similar to the target RNA modified to render it resistant to miRNA induced cleavage, e.g. by modifying the sequence thereof such that a variation is introduced in the nucleotide of the target sequence complementary to the nucleotides 10 or 11 of the miRNA resulting in a mismatch.
  • a microRNA-resistant target may be implemented.
  • a silent mutation may be introduced in the microRNA binding site of the target gene so that the DNA and resulting RNA sequences are changed in a way that prevents microRNA binding, but the amino acid sequence of the protein is unchanged.
  • a new sequence can be synthesized instead of the existing binding site, in which the DNA sequence is changed, resulting in lack of miRNA binding to its target.
  • Tables 10 and 11 below provide non-limiting examples of target mimics and target resistant sequences that can be used to down-regulate the activity of the miRs of the invention.
  • the target mimic or micro-RNA resistant target is linked to the promoter naturally associated with the pre-miRNA recognizing the target gene and introduced into the plant cell.
  • the miRNA target mimic or micro-RNA resistant target RNA will be expressed under the same circumstances as the miRNA and the target mimic or micro-RNA resistant target RNA will substitute for the non-target mimic/micro-RNA resistant target RNA degraded by the miRNA induced cleavage.
  • Non-functional miRNA alleles or miRNA resistant target genes may also be introduced by homologous recombination to substitute the miRNA encoding alleles or miRNA sensitive target genes.
  • Recombinant expression is effected by cloning the nucleic acid of interest (e.g., miRNA, target gene, silencing agent etc) into a nucleic acid expression construct under the expression of a plant promoter, as further described hereinbelow.
  • nucleic acid of interest e.g., miRNA, target gene, silencing agent etc
  • a miRNA inhibitor is typically between about 17 to 25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90 % complementary to the 5' to 3' sequence of a mature miRNA.
  • a miRNA inhibitor molecule is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein.
  • a miRNA inhibitor has a sequence (from 5' to 3 * ) that is or is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100 % complementary, or any range derivable therein, to the 5' to 3' sequence of a mature miRNA, particularly a mature, naturally occurring miRNA.
  • the present inventors While further reducing the present invention to practice, the present inventors have identified gene targets for the differentially expressed miRNA molecules. It is therefore contemplated, that gene targets of those miRNAs that are down regulated during stress should be overexpressed in order to confer tolerance, while gene targets of those miR As that are up regulated during stress should be downregulated in the plant in order to confer tolerance.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 82 %, 84 %, 85 %, 86 %, 88 %, 90 %, 92 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % homologous to SEQ ID NOs: 927-1021 (gene targets of down regulated miRNAs, see Table 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • Nucleic acid sequences (also referred to herein as polynucleotides) of the polypeptides of some embodiments of the invention may be optimized for expression in a specific plant host. Examples of such sequence modifications include, but are not limited to, an altered G/C content to more closely approach that typically found in the plant species of interest, and the removal of codons atypically found in the plant species commonly referred to as codon optimization.
  • an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to utilize statistically-preferred or statistically-favored codons within the plant.
  • the nucleotide sequence typically is examined at the DNA level and the coding region optimized for expression in the plant species determined using any suitable procedure, for example as described in Sardana et al. (1996, Plant Cell Reports 15:677-681).
  • the standard deviation of codon usage may be calculated by first finding the squared proportional deviation of usage of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the average squared deviation.
  • a table of codon usage from highly expressed genes of dicotyledonous plants is compiled using the data of Murray et al. (1989, Nuc Acids Res. 17:477-498).
  • Codon Usage Database contains codon usage tables for a number of different species, with each codon usage table having been statistically determined based on the data present in Genbank.
  • a naturally- occurring nucleotide sequence encoding a protein of interest can be codon optimized for that particular plant species. This is effected by replacing codons that may have a low statistical incidence in the particular species genome with corresponding codons, in regard to an amino acid, that are statistically more favored.
  • one or more less- favored codons may be selected to delete existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends to add signal peptide or termination cassettes, internal sites that might be used to cut and splice segments together to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively effect mRNA stability or expression.
  • codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically-favored with regards to a particular plant, and modifying these codons in accordance with a codon usage table of the particular plant to produce a codon optimized derivative.
  • a modified nucleotide sequence may be fully or partially optimized for plant codon usage provided that the protein encoded by the modified nucleotide sequence is produced at a level higher than the protein encoded by the corresponding naturally occurring or native gene. Construction of synthetic genes by altering the codon usage is described in for example PCT Patent Application 93/07278.
  • Target genes which are contemplated according to the present teachings are provided in the polynucleotide sequences which comprise nucleic acid sequences as set forth in the maize polynucleotides listed in Tables 5 and 6).
  • the present teachings also relate to orthologs or homologs at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, or at least about 95 % or more identical or similar to SEQ ID NO: 895-926 or 1022-1090 (polynucleotides listed in Tables 5 and 6). Parameters for determining the level of identity are provided hereinbelow.
  • target genes which are contemplated according to the present teachings are provided in the polypeptide sequences which comprise amino acid sequences as set forth the maize polypeptides of Tables 5 and 6).
  • the present teachings also relate to of orthologs or homologs at least about 60 %, at least about 65 %, at least about 70 %, at least about 75 %, at least about 80 %, at least about 85 %, at least about 90 %, or at least about 95 % or more identical or similar to SEQ ID NO: 854-894 or 927-1021 (Tables 5 and 6).
  • Homology e.g., percent homology, identity + similarity
  • Homology comparison software including for example, the TBLASTN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters, when starting from a polypeptide sequence; or the tBLASTX algorithm (available via the NCBI) such as by using default parameters, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database.
  • NCBI National Center of Biotechnology Information
  • tBLASTX algorithm available via the NCBI
  • homologous refers to identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences.
  • Homologous sequences include both orthologous and paralogous sequences.
  • the term "paralogous” relates to gene-duplications within the genome of a species leading to paralogous genes.
  • the term “orthologous” relates to homologous genes in different organisms due to ancestral relationship.
  • One option to identify orthologues in monocot plant species is by performing a reciprocal blast search. This may be done by a first blast involving blasting the sequence-of-interest against any sequence database, such as the publicly available NCBI database which may be found at: Hypertext Transfer Protocol ://World Wide Web (dot) ncbi (dot) nlm (dot) nih (dot) gov. The blast results may be filtered.
  • the ClustalW program may be used [Hypertext Transfer Protocol ://World Wide Web (dot) ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighbor-joining tree (Hypertext Transfer Protocol://en (dot) wikipedia (dot) org/wiki/Neighbor-joining) which helps visualizing the clustering.
  • genes which down- regulation may be done in order to improve their NUE, biomass, vigor, yield and abiotic stress tolerance.
  • a method of improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant comprising expressing within the plant an exogenous polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 %, or 100 % homologous to SEQ ID NOs: 854- 894 (polypeptides of Table 5), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant, thereby improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of the plant.
  • Down regulation of activity or expression is by at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or even complete (100 %) loss of activity or expression.
  • Assays for measuring gene expression can be effected at the protein level (e.g,. Western blot, ELISA) or at the mRNA level such as by RT-PCR.
  • the amino acid sequence of the target gene is as set forth in SEQ ID NOs: 854-894 of Table 5.
  • amino acid sequence of the target gene is encoded by a polynucleotide sequence as set forth in SEQ ID NOs: 895-926 of Table 5.
  • polynucleotide downregulating agents that inhibit (also referred to herein as inhibitors or nucleic acid agents) the expression of a target gene are given below.
  • any of these methods when specifically referring to downregulating expression/activity of the target genes can be used, at least in part, to downregulate expression or activity of endogenous RNA molecules,
  • inhibition of the expression of target gene may be obtained by sense suppression or cosuppression.
  • an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding a target gene in the "sense" orientation. Over-expression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show the greatest inhibition of target gene expression.
  • the polynucleotide used for cosuppression may correspond to all or part of the sequence encoding the target gene, all or part of the 5' and/or 3' untranslated region of a target transcript, or all or part of both the coding sequence and the untranslated regions of a transcript encoding the target gene.
  • the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed.
  • Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin, et al, (2002) Plant Cell 15: 1517-1532. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell, et al, (1995) Proc. Natl. Acad. Sci. USA 91 :3590-3596; Jorgensen, et al, (1996) Plant Mol. Biol. 31 :957-973; Johansen and Carrington, (2001) Plant Physiol.
  • nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, optimally greater than about 65 % sequence identity, more optimally greater than about 85 % sequence identity, most optimally greater than about 95 % sequence identity. See, U.S. Pat. Nos. 5,283,185 and 5,035,323; herein incorporated by reference.
  • Transcriptional gene silencing may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing.
  • inhibition of the expression of the target gene may be obtained by antisense suppression.
  • the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the target gene. Over-expression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of target gene expression.
  • the polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the target gene, all or part of the complement of the 5' and/or 3' untranslated region of the target gene transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the target gene.
  • the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence.
  • Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant.
  • portions of the antisense nucleotides may be used to disrupt the expression of the target gene.
  • sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, 300, 500, 550, 500, 550 or greater may be used.
  • Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu, et al, (2002) Plant Physiol. 129: 1732-1753 and U.S. Pat. No. 5,759,829, which is herein incorporated by reference.
  • Efficiency of antisense suppression may be increased by including a poly-dt region in the expression cassette at a position 3' to the antisense sequence and 5' of the polyadenylation signal. See, US Patent Publication Number 20020058815.
  • inhibition of the expression of a target gene may be obtained by double-stranded RNA (dsRNA) interference.
  • dsRNA interference a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.
  • Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of target gene expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse, et al, (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13965, Liu, et al, (2002) Plant Physiol. 129: 1732-1753, and WO 99/59029, WO 99/53050, WO 99/61631, and WO 00/59035;
  • inhibition of the expression of one or more target gene may be obtained by hairpin RNA (hpRNA) interference or intron- containing hairpin RNA (ihpRNA) interference.
  • hpRNA hairpin RNA
  • ihpRNA intron- containing hairpin RNA
  • the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single- stranded loop region and a base-paired stem.
  • the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence.
  • the base-paired stem region of the molecule generally determines the specificity of the RNA interference.
  • hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl. Acad.
  • the interfering molecules have the same general structure as for hpRNA, but the RNA molecule additionally comprises an intron that is capable of being spliced in the cell in which the ihpRNA is expressed.
  • the use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith, et al, (2000) Nature 507:319-320. In fact, Smith, et al, show 100 % suppression of endogenous gene expression using ihpRNA-mediated interference.
  • the expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA.
  • the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene.
  • it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00905, herein incorporated by reference.
  • Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus.
  • the viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication.
  • the transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for target gene).
  • Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe, (1997) EMBO J. 16:3675-3685, Angell and Baulcombe, (1999) Plant J. 20:357-362, and U.S. Pat. No. 6,656,805, each of which is herein incorporated by reference.
  • the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of target gene.
  • the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the target gene. This method is described, for example, in U.S. Pat. No. 5,987,071, herein incorporated by reference.
  • the activity of a miRNA or a target gene is reduced or eliminated by disrupting the gene encoding the target polypeptide.
  • the gene encoding the target polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have reduced response regulator activity.
  • nucleic acid agents described herein for overexpression or downregulation of either the target gene of the miR A
  • nucleic acid construct comprising a nucleic acid sequence encoding a the nucleic acid agent (e.g., miRNA or a precursor thereof as described herein, gene targetm or silencing agent), said nucleic acid sequence being under a transcriptional control of a regulatory sequence such as a tissue specific promoter.
  • a the nucleic acid agent e.g., miRNA or a precursor thereof as described herein, gene targetm or silencing agent
  • An exemplary nucleic acid construct which can be used for plant transformation include, the pORE E2 binary vector ( Figure 1) in which the relevant nucleic acid sequence is ligated under the transcriptional control of a promoter.
  • a coding nucleic acid sequence is "operably linked” or “transcriptionally linked to a regulatory sequence (e.g., promoter)" if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto.
  • a regulatory sequence e.g., promoter
  • the regulatory sequence controls the transcription of the miRNA or precursor thereof, gene target or silencing agent.
  • regulatory sequence means any DNA, that is involved in driving transcription and controlling (i.e., regulating) the timing and level of transcription of a given DNA sequence, such as a DNA coding for a miRNA, precursor or inhibitor of same.
  • a 5' regulatory region is a DNA sequence located upstream (i.e., 5') of a coding sequence and which comprises the promoter and the 5 '-untranslated leader sequence.
  • a 3' regulatory region is a DNA sequence located downstream (i.e., 3') of the coding sequence and which comprises suitable transcription termination (and/or regulation) signals, including one or more polyadenylation signals.
  • the promoter is a plant-expressible promoter.
  • the term "plant-expressible promoter” means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin. Thus, any suitable promoter sequence can be used by the nucleic acid construct of the present invention. According to some embodiments of the invention, the promoter is a constitutive promoter, a tissue-specific promoter or an inducible promoter (e.g. an abiotic stress-inducible promoter).
  • Suitable constitutive promoters include, for example, hydroperoxide lyase (HPL) promoter, CaMV 35S promoter (Odell et al, Nature 313:810-812, 1985); Arabidopsis At6669 promoter (see PCT Publication No. WO04081173A2); Arabidopsis new At6669 promoter; maize Ubi 1 (Christensen et al., Plant Sol. Biol. 18:675-689, 1992); rice actin (McElroy et al, Plant Cell 2:163-171, 1990); pEMU (Last et al, Theor. Appl. Genet.
  • HPL hydroperoxide lyase
  • CaMV 35S promoter Odell et al, Nature 313:810-812, 1985
  • Arabidopsis At6669 promoter see PCT Publication No. WO04081173A2
  • Arabidopsis new At6669 promoter maize Ubi 1 (
  • tissue-specific promoters include, but not limited to, leaf-specific promoters [such as described, for example, by Yamamoto et al, Plant J. 12:255-265, 1997; Kwon et al, Plant Physiol. 105:357-67, 1994; Yamamoto et al, Plant Cell Physiol. 35:773-778, 1994; Gotor et al, Plant J. 3:509-18, 1993; Orozco et al, Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al, Proc. Natl. Acad. Sci.
  • seed-preferred promoters e.g., from seed specific genes (Simon, et al, Plant Mol. Biol. 5. 191, 1985; Scofield, et al, J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), Brazil Nut albumin (Pearson' et al., Plant Mol. Biol. 18: 235- 245, 1992), legumin (Ellis, et al. Plant Mol. Biol. 10: 203- 214, 1988), Glutelin (rice) (Takaiwa, et al, Mol. Gen. Genet.
  • endosperm specific promoters e.g., wheat LMW and HMW, glutenin-1 (Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat a, b and g gliadins (EMB03: 1409-15, 1984), Barley ltrl promoter, barley Bl, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750- 60, 1996), Barley DOF (Mena et al, The Plant Journal, 116(1): 53- 62, 1998), Biz2 (EP99106056.7), Synthetic promoter (Vicente-Carbajosa et al, Plant J.
  • flower-specific promoters e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al, Mol. Gen Genet. 217:240-245; 1989), apetala- 3].
  • root-specific promoters such as the ROOTP promoter described in Vissenberg K, et al. Plant Cell Physiol. 2005 January; 46(1): 192- 200.
  • the nucleic acid construct of some embodiments of the invention can further include an appropriate selectable marker and/or an origin of replication.
  • the nucleic acid construct of some embodiments of the invention can be utilized to stably or transiently transform plant cells.
  • stable transformation the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait.
  • transient transformation the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • the polynucleotides may be synthesized using any method known in the art, including either enzymatic syntheses or solid-phase syntheses. These are especially useful in the case of short polynucleotide sequences with or without modifications as explained above.
  • Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example: Sambrook, J. and Russell, D. W. (2001), "Molecular Cloning: A Laboratory Manual”; Ausubel, R. M.
  • Agrobacterium-mediated gene transfer e.g., T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes
  • Agrobacterium-mediated gene transfer see for example, Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2- 25; Gatenby, in Plant Biotechnology, eds. Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
  • the Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
  • the exogenous polynucleotide is introduced into the plant by infecting the plant with a bacteria, such as using a floral dip transformation method (as described in further detail in Example 5, of the Examples section which follows).
  • DNA transfer into plant cells There are various methods of direct DNA transfer into plant cells.
  • electroporation the protoplasts are briefly exposed to a strong electric field.
  • microinjection the DNA is mechanically injected directly into the cells using very small micropipettes.
  • microparticle bombardment the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated into cells or plant tissues.
  • Micropropagation is a process of growing new generation plants from a single piece of tissue that has been excised from a selected parent plant or cultivar.
  • the new generation plants which are produced are genetically identical to, and have all of the characteristics of, the original plant.
  • Micropropagation allows mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in the preservation of the characteristics of the original transgenic or transformed plant.
  • the advantages of cloning plants are the speed of plant multiplication and the quality and uniformity of plants produced.
  • Micropropagation is a multi-stage procedure that requires alteration of culture medium or growth conditions between stages.
  • the micropropagation process involves four basic stages: Stage one, initial tissue culturing; stage two, tissue culture multiplication; stage three, differentiation and plant formation; and stage four, greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • stage two tissue culture multiplication
  • stage three differentiation and plant formation
  • stage four greenhouse culturing and hardening.
  • stage one initial tissue culturing
  • the tissue culture is established and certified contaminant- free.
  • stage two the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production goals.
  • stage three the tissue samples grown in stage two are divided and grown into individual plantlets.
  • the transformed plantlets are transferred to a greenhouse for hardening where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.
  • transient transformation of leaf cells, meristematic cells or the whole plant is also envisaged by the present invention.
  • Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
  • Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, Tobacco mosaic virus (TMV), brome mosaic virus (BMV) and Bean Common Mosaic Virus (BV or BCMV). Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988).
  • TMV Tobacco mosaic virus
  • BMV brome mosaic virus
  • BV or BCMV Bean Common Mosaic Virus Transformation of plants using plant viruses is described in U.S. Pat. No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-
  • the virus used for transient transformations is avirulent and thus is incapable of causing severe symptoms such as reduced growth rate, mosaic, ring spots, leaf roll, yellowing, streaking, pox formation, tumor formation and pitting.
  • a suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus.
  • Virus attenuation may be effected by using methods well known in the art including, but not limited to, sub-lethal heating, chemical treatment or by directed mutagenesis techniques such as described, for example, by Kurihara and Watanabe (Molecular Plant Pathology 4:259- 269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
  • Suitable virus strains can be obtained from available sources such as, for example, the American Type culture Collection (ATCC) or by isolation from infected plants. Isolation of viruses from infected plant tissues can be effected by techniques well known in the art such as described, for example by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. Briefly, tissues of an infected plant believed to contain a high concentration of a suitable virus, preferably young leaves and flower petals, are ground in a buffer solution (e.g., phosphate buffer solution) to produce a virus infected sap which can be used in subsequent inoculations.
  • a buffer solution e.g., phosphate buffer solution
  • the virus When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with the foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coat proteins which will encapsidate the viral DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translation of the viral genes to produce the coat protein(s) which encapsidate the viral RNA.
  • a plant viral nucleic acid in which the native coat protein coding sequence has been deleted from a viral nucleic acid, a non-native plant viral coat protein coding sequence and a non-native promoter, preferably the subgenomic promoter of the non-native coat protein coding sequence, capable of expression in the plant host, packaging of the recombinant plant viral nucleic acid, and ensuring a systemic infection of the host by the recombinant plant viral nucleic acid, has been inserted.
  • the coat protein gene may be inactivated by insertion of the non-native nucleic acid sequence within it, such that a protein is produced.
  • the recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters.
  • Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and incapable of recombination with each other and with native subgenomic promoters.
  • Non-native (foreign) nucleic acid sequences may be inserted adjacent the native plant viral subgenomic promoter or the native and a non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included.
  • the non-native nucleic acid sequences are transcribed or expressed in the host plant under control of the subgenomic promoter to produce the desired products.
  • a recombinant plant viral nucleic acid is provided as in the first embodiment except that the native coat protein coding sequence is placed adjacent one of the non-native coat protein subgenomic promoters instead of a non- native coat protein coding sequence.
  • a recombinant plant viral nucleic acid in which the native coat protein gene is adjacent its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid.
  • the inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are incapable of recombination with each other and with native subgenomic promoters.
  • Non-native nucleic acid sequences may be inserted adjacent the non-native subgenomic plant viral promoters such that the sequences are transcribed or expressed in the host plant under control of the subgenomic promoters to produce the desired product.
  • a recombinant plant viral nucleic acid is provided as in the third embodiment except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
  • the viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus.
  • the recombinant plant viral nucleic acid or recombinant plant virus is used to infect appropriate host plants.
  • the recombinant plant viral nucleic acid is capable of replication in the host, systemic spread in the host, and transcription or expression of foreign gene(s) (isolated nucleic acid) in the host to produce the desired sequence.
  • nucleic acid molecule of the present invention can also be introduced into a chloroplast genome thereby enabling chloroplast expression.
  • a technique for introducing exogenous nucleic acid sequences to the genome of the chloroplasts involves the following procedures. First, plant cells are chemically treated so as to reduce the number of chloroplasts per cell to about one. Then, the exogenous nucleic acid is introduced via particle bombardment into the cells with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplasts. The exogenous nucleic acid is selected such that it is integratable into the chloroplast's genome via homologous recombination which is readily effected by enzymes inherent to the chloroplast.
  • the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid stretch which is derived from the chloroplast's genome.
  • the exogenous nucleic acid includes a selectable marker, which serves by sequential selection procedures to ascertain that all or substantially all of the copies of the chloroplast genomes following such selection will include the exogenous nucleic acid. Further details relating to this technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507 which are incorporated herein by reference.
  • the present invention also contemplates a transgenic plant exogenous ly expressing the polynucleotide/nucleic acid agent of the invention.
  • the transgenic plant exogenously expresses a polynucleotide having a nucleic acid sequence at least , 80 %, 85 %, 90 %, 95 % or even 100 % identical to SEQ ID NOs: 2-20, 23-37, 57-449, 21-22, 38-52, 1209, 1211, 1212, 454-846 and 53-56, 1209 (Tables 1, 3 and 4), wherein said nucleic acid sequence is capable of regulating nitrogen use efficiency of the plant.
  • the exogenous polynucleotide encodes a precursor of said nucleic acid sequence.
  • the stem-loop precursor is at least 60 %, 65 % , 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or even 100 % identical to SEQ ID NOs: 21-22, 38-52, 1209, 121 1, 1212, 454-846, 53-56, 1209 (Tables 1, 3 and 4) identical to SEQ ID NO: 21-22, 38-52, 1209, 1211, 1212, 54-846 and 53-56, 1209 (precursor sequences of Tables 1, 3 and 4). More specifically the exogenous polynucleotide is selected from the group consisting of SEQ ID NO: 21-22 and 38-52, 1209, 1211, 1212 (precursor and mature sequences of upregulated Tables 1 and 3).
  • transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a gene encoding a miRNA molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209 (downregulated Tables 1 and 4) or homologs thereof which are at least at least 80 %, 85 %, 90 % or 95 % identical to SEQ ID NOs: 4, 1-3, 5, 57-449, 454-846 and 53-56, 1209 (downregulated Tables 1 and 4) ⁇
  • transgenic plant expresses the nucleic acid agent of Tables
  • transgenic plant expresses the nucleic acid agent of Tables 8 and 11.
  • transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 854-894 (polypeptides of Table 5), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.
  • transgenic plant exogenously expressing a polynucleotide encoding a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 927-1021 (polypeptides of Table 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.
  • transgenic plant exogenously expressing a polynucleotide which downregulates an activity or expression of a polypeptide having an amino acid sequence at least 80 %, 85 %, 90 %, 95 % or even 100 % homologous to SEQ ID NOs: 854-894, 927-1021 (targets of Tables 5 and 6), wherein said polypeptide is capable of regulating nitrogen use efficiency of the plant.
  • hybrid plants refers to a plant or a part thereof resulting from a cross between two parent plants, wherein one parent is a genetically engineered plant of the invention (transgenic plant expressing an exogenous miRNA sequence or a precursor thereof). Such a cross can occur naturally by, for example, sexual reproduction, or artificially by, for example, in vitro nuclear fusion. Methods of plant breeding are well-known and within the level of one of ordinary skill in the art of plant biology.
  • the invention also envisages expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve superior effect on the efficiency of nitrogen use, yield, vigor and biomass of the plant.
  • Expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing multiple nucleic acid constructs, each including a different exogenous polynucleotide, into a single plant cell.
  • the transformed cell can then be regenerated into a mature plant using the methods described hereinabove.
  • expressing a plurality of exogenous polynucleotides in a single host plant can be effected by co-introducing into a single plant-cell a single nucleic-acid construct including a plurality of different exogenous polynucleotides.
  • Such a construct can be designed with a single promoter sequence which can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences.
  • the construct can include several promoter sequences each linked to a different exogenous polynucleotide sequence.
  • the plant cell transformed with the construct including a plurality of different exogenous polynucleotides can be regenerated into a mature plant, using the methods described hereinabove.
  • expressing a plurality of exogenous polynucleotides can be effected by introducing different nucleic acid constructs, including different exogenous polynucleotides, into a plurality of plants.
  • the regenerated transformed plants can then be cross-bred and resultant progeny selected for superior yield or tolerance traits as described above, using conventional plant breeding techniques.
  • Expression of the miR As of the present invention or precursors thereof can be qualified using methods which are well known in the art such as those involving gene amplification e.g., PCR or RT-PCR or Northern blot or in-situ hybrdization.
  • the plant expressing the exogenous polynucleotide(s) is grown under stress (nitrogen or abiotic) or normal conditions (e.g., biotic conditions and/or conditions with sufficient water, nutrients such as nitrogen and fertilizer).
  • stress nitrogen or abiotic
  • normal conditions e.g., biotic conditions and/or conditions with sufficient water, nutrients such as nitrogen and fertilizer.
  • the method further comprises growing the plant expressing the exogenous polynucleotide(s) under abiotic stress or nitrogen limiting conditions.
  • abiotic stress conditions include, water deprivation, drought, excess of water (e.g., flood, waterlogging), freezing, low temperature, high temperature, strong winds, heavy metal toxicity, anaerobiosis, nutrient deficiency, nutrient excess, salinity, atmospheric pollution, intense light, insufficient light, or UV irradiation, etiolation and atmospheric pollution.
  • the invention encompasses plants exogenously expressing the polynucleotide(s), the nucleic acid constructs of the invention.
  • RNA-m situ hybridization Methods of determining the level in the plant of the RNA transcribed from the exogenous polynucleotide are well known in the art and include, for example, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-m situ hybridization.
  • RT-PCR reverse transcription polymerase chain reaction
  • sub-sequence data of those polynucleotides described above can be used as markers for marker assisted selection (MAS), in which a marker is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g., tolerance to abiotic stress).
  • MAS marker assisted selection
  • Nucleic acid data of the present teachings may contain or be linked to polymorphic sites or genetic markers on the genome such as restriction fragment length polymorphism (RFLP), microsatellites and single nucleotide polymorphism (SNP), DNA fingerprinting (DFP), amplified fragment length polymorphism (AFLP), expression level polymorphism, and any other polymorphism at the DNA or RNA sequence.
  • RFLP restriction fragment length polymorphism
  • SNP single nucleotide polymorphism
  • DFP DNA fingerprinting
  • AFLP amplified fragment length polymorphism
  • expression level polymorphism any other polymorphism at the DNA or RNA sequence.
  • marker assisted selections include, but are not limited to, selection for a morphological trait (e.g., a gene that affects form, coloration, male sterility or resistance such as the presence or absence of awn, leaf sheath coloration, height, grain color, aroma of rice); selection for a biochemical trait (e.g., a gene that encodes a protein that can be extracted and observed; for example, isozymes and storage proteins); selection for a biological trait (e.g., pathogen races or insect biotypes based on host pathogen or host parasite interaction can be used as a marker since the genetic constitution of an organism can affect its susceptibility to pathogens or parasites).
  • a morphological trait e.g., a gene that affects form, coloration, male sterility or resistance such as the presence or absence of awn, leaf sheath coloration, height, grain color, aroma of rice
  • selection for a biochemical trait e.g., a gene that encodes a protein that
  • polynucleotides described hereinabove can be used in a wide range of economical plants, in a safe and cost effective manner.
  • Plant lines exogenously expressing the polynucleotide of the invention can be screened to identify those that show the greatest increase of the desired plant trait.
  • a method of evaluating a trait of a plant comprising: (a) expressing in a plant or a portion thereof the nucleic acid construct; and (b) evaluating a trait of a plant as compared to a wild type plant of the same type; thereby evaluating the trait of the plant.
  • the effect of the transgene (the exogenous polynucleotide) on different plant characteristics may be determined any method known to one of ordinary skill in the art.
  • tolerance to limiting nitrogen conditions may be compared in transformed plants ⁇ i.e., expressing the transgene) compared to non-transformed (wild type) plants exposed to the same stress conditions ( other stress conditions are contemplated as well, e.g. water deprivation, salt stress e.g. salinity, suboptimal temperatureosmotic stress, and the like), using the following assays.
  • Fertilizer use efficiency To analyze whether the transgenic plants are more responsive to fertilizers, plants are grown in agar plates or pots with a limited amount of fertilizer, as described, for example, in Yanagisawa et al (Proc Natl Acad Sci U S A. 2004; 101 :7833-8). The plants are analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain. The parameters checked are the overall size of the mature plant, its wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant.
  • NUE nitrogen use efficiency
  • PUE phosphate use efficiency
  • KUE potassium use efficiency
  • Nitrogen use efficiency To analyze whether the transgenic plants (e.g., Arabidopsis plants) are more responsive to nitrogen, plant are grown in 0.75-3 millimolar (mM, nitrogen deficient conditions) or 10, 6-9 mM (optimal nitrogen concentration). Plants are allowed to grow for additional 25 days or until seed production. The plants are then analyzed for their overall size, time to flowering, yield, protein content of shoot and/or grain/ seed production. The parameters checked can be the overall size of the plant, wet and dry weight, the weight of the seeds yielded, the average seed size and the number of seeds produced per plant.
  • mM nitrogen deficient conditions
  • 10-9 mM optimal nitrogen concentration
  • Nitrogen Use efficiency assay using plantlets - The assay is done according to Yanagisawa-S. et al. with minor modifications ("Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low- nitrogen conditions" Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, transgenic plants which are grown for 7-10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogen-limiting conditions: MS media in which the combined nitrogen concentration (NH 4 NO3 and KNO3) was 0.75 mM (nitrogen deficient conditions) or 6-15 mM (optimal nitrogen concentration).
  • Plants are allowed to grow for additional 30-40 days and then photographed, individually removed from the Agar (the shoot without the roots) and immediately weighed (fresh weight) for later statistical analysis. Constructs for which only Tl seeds are available are sown on selective media and at least 20 seedlings (each one representing an independent transformation event) are carefully transferred to the nitrogen- limiting media. For constructs for which T2 seeds are available, different transformation events are analyzed. Usually, 20 randomly selected plants from each event are transferred to the nitrogen- limiting media allowed to grow for 3-4 additional weeks and individually weighed at the end of that period. Transgenic plants are compared to control plants grown in parallel under the same conditions. Mock- transgenic plants expressing the uidA reporter gene (GUS) under the same promoter or transgenic plants carrying the same promoter but lacking a reporter gene are used as control.
  • GUS uidA reporter gene
  • N (nitrogen) concentration determination in the structural parts of the plants involves the potassium persulfate digestion method to convert organic N to NO3 " (Purcell and King 1996 Argon. J. 88: 111-113, the modified Cd " mediated reduction of N0 3 to N0 2 (Vodovotz 1996 Biotechniques 20:390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). The absorbance values are measured at 550 nm against a standard curve of NaN0 2 . The procedure is described in details in Samonte et al. 2006 Agron. J. 98: 168-176.
  • Tolerance to abiotic stress can be evaluated by determining the differences in physiological and/or physical condition, including but not limited to, vigor, growth, size, or root length, or specifically, leaf color or leaf area size of the transgenic plant compared to a non-modified plant of the same species grown under the same conditions.
  • Other techniques for evaluating tolerance to abiotic stress include, but are not limited to, measuring chlorophyll fluorescence, photosynthetic rates and gas exchange rates. Further assays for evaluating tolerance to abiotic stress are provided hereinbelow and in the Examples section which follows.
  • Drought tolerance assay - Soil-based drought screens are performed with plants overexpressing the polynucleotides detailed above. Seeds from control Arabidopsis plants, or other transgenic plants overexpressing nucleic acid of the invention are germinated and transferred to pots. Drought stress is obtained after irrigation is ceased. Transgenic and control plants are compared to each other when the majority of the control plants develop severe wilting. Plants are re-watered after obtaining a significant fraction of the control plants displaying a severe wilting. Plants are ranked comparing to controls for each of two criteria: tolerance to the drought conditions and recovery (survival) following re-watering.
  • Quantitative parameters of tolerance measured include, but are not limited to, the average wet and dry weight, growth rate, leaf size, leaf coverage (overall leaf area), the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as drought stress tolerant plants
  • Salinity tolerance assay - Transgenic plants with tolerance to high salt concentrations are expected to exhibit better germination, seedling vigor or growth in high salt.
  • Salt stress can be effected in many ways such as, for example, by irrigating the plants with a hyperosmotic solution, by cultivating the plants hydroponically in a hyperosmotic growth solution (e.g., Hoagland solution with added salt), or by culturing the plants in a hyperosmotic growth medium [e.g., 50 % Murashige-Skoog medium (MS medium) with added salt].
  • a hyperosmotic growth medium e.g., 50 % Murashige-Skoog medium (MS medium) with added salt.
  • the salt concentration in the irrigation water, growth solution, or growth medium can be adjusted according to the specific characteristics of the specific plant cultivar or variety, so as to inflict a mild or moderate effect on the physiology and/or morphology of the plants (for guidelines as to appropriate concentration see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002, and reference therein).
  • a salinity tolerance test can be performed by irrigating plants at different developmental stages with increasing concentrations of sodium chloride (for example 50 mM, 150 mM, 300 mM NaCl) applied from the bottom and from above to ensure even dispersal of salt. Following exposure to the stress condition the plants are frequently monitored until substantial physiological and/or morphological effects appear in wild type plants. Thus, the external phenotypic appearance, degree of chlorosis and overall success to reach maturity and yield progeny are compared between control and transgenic plants. Quantitative parameters of tolerance measured include, but are not limited to, the average wet and dry weight, growth rate, leaf size, leaf coverage (overall leaf area), the weight of the seeds yielded, the average seed size and the number of seeds produced per plant. Transformed plants not exhibiting substantial physiological and/or morphological effects, or exhibiting higher biomass than wild-type plants, are identified as abiotic stress tolerant plants.
  • sodium chloride for example 50 mM, 150 mM, 300 mM NaCl
  • Osmotic tolerance test Osmotic stress assays (including sodium chloride and PEG assays) are conducted to determine if an osmotic stress phenotype was sodium chloride-specific or if it was a general osmotic stress related phenotype. Plants which are tolerant to osmotic stress may have more tolerance to drought and/or freezing. For salt and osmotic stress experiments, the medium is supplemented for example with 50 mM, 100 mM, 200 mM NaCl or 15 %, 20 % or 25 % PEG.
  • Cold stress tolerance One way to analyze cold stress is as follows. Mature (25 day old) plants are transferred to 4 °C chambers for 1 or 2 weeks, with constitutive light. Later on plants are moved back to greenhouse. Two weeks later damages from chilling period, resulting in growth retardation and other phenotypes, are compared between control and transgenic plants, by measuring plant weight (wet and dry), and by comparing growth rates measured as time to flowering, plant size, yield, and the like.
  • Heat stress tolerance One way to measure heat stress tolerance is by exposing the plants to temperatures above 34 °C for a certain period. Plant tolerance is examined after transferring the plants back to 22 °C for recovery and evaluation after 5 days relative to internal controls (non-transgenic plants) or plants not exposed to neither cold or heat stress.
  • plant vigor can be calculated by the increase in growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight, oil content, seed yield and the like per time.
  • increased yield of rice can be manifested by an increase in one or more of the following: number of plants per growing area, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight (1000-weight), increase oil content per seed, increase starch content per seed, among others.
  • An increase in yield may also result in modified architecture, or may occur because of modified architecture.
  • increased yield of soybean may be manifested by an increase in one or more of the following: number of plants per growing area, number of pods per plant, number of seeds per pod, increase in the seed filling rate, increase in thousand seed weight (1000-weight), reduce pod shattering, increase oil content per seed, increase protein content per seed, among others.
  • An increase in yield may also result in modified architecture, or may occur because of modified architecture.
  • the present invention is of high agricultural value for increasing tolerance of plants to nitrogen deficiency or abiotic stress as well as promoting the yield, biomass and vigor of commercially desired crops.
  • a food or feed comprising the plants or a portion thereof of the present invention.
  • the transgenic plants of the present invention or parts thereof are comprised in a food or feed product (e.g., dry, liquid, paste).
  • a food or feed product is any ingestible preparation containing the transgenic plants, or parts thereof, of the present invention, or preparations made from these plants.
  • the plants or preparations are suitable for human (or animal) consumption, i.e. the transgenic plants or parts thereof are more readily digested.
  • Feed products of the present invention further include a oil or a beverage adapted for animal consumption. It will be appreciated that the transgenic plants, or parts thereof, of the present invention may be used directly as feed products or alternatively may be incorporated or mixed with feed products for consumption. Furthermore, the food or feed products may be processed or used as is.
  • Exemplary feed products comprising the transgenic plants, or parts thereof include, but are not limited to, grains, cereals, such as oats, e.g. black oats, barley, wheat, rye, sorghum, corn, vegetables, leguminous plants, especially soybeans, root vegetables and cabbage, or green forage, such as grass or hay.
  • oats e.g. black oats, barley, wheat, rye, sorghum, corn, vegetables, leguminous plants, especially soybeans, root vegetables and cabbage, or green forage, such as grass or hay.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • At least one compound may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • Corn seeds were obtained from Galil seeds (Israel). Corn variety 5605 was used in all experiments. Plants were grown at 28 °C under a 16 hr light:8 hr dark regime.
  • Corn seeds were germinated and grown on defined growth media containing either sufficient (100% N 2 ) or insufficient nitrogen levels (1 % or 10 % N 2 ). Seedlings aged one or two weeks were used for tissue samples for RNA analysis, as described below.
  • RNA of leaf or root samples from four to eight biological repeats were extracted using the mirVanaTM kit (Ambion, Austin, TX) by pooling 3-4 plants to one biological repeat.
  • Custom microarrays were manufactured by Agilent Technologies by in situ synthesis.
  • the first generation microarray consisted of a total of 13619 non-redundant DNA probes, the majority of which arose from deep sequencing data and includes different small RNA molecules (i.e. miRNAs, siRNA and predicted small RNA sequences), with each probe being printed once.
  • An in-depth analysis of the first generation microarray which included hybridization experiments as well as structure and orientation verifications on all its small RNAs, resulted in the formation of an improved, second generation, microarray.
  • Wild type maize plants were allowed to grow at standard, optimal conditions or nitrogen deficient conditions for one or two weeks, at the end of which they were evaluated for NUE. Three to four plants from each group were used for reproducibility. Four to eight repeats were obtained for each group, and RNA was extracted from leaf or root tissue. The expression level of the maize miRNAs was analyzed by high throughput microarray to identify miRNAs that were differentially expressed between the experimental groups.
  • Tables 1-2 below presents sequences that were found to be differentially expressed in corn grown in various nitrogen levels. To clarify, the sequence of an up- regulated miRNA is induced under nitrogen limiting conditions and the sequence of a down-regulated miRNA is repressed under nitrogen limiting conditions compared to optimal conditions. Table 1: Differentially Expressed miRNAs in Leaf of Plants Growing under
  • the small RNA sequences of the invention that were either down- or upregulated under nitrogen limiting conditions were examined for homologous and orthologous sequences using the miRBase database (wwwdotmirbasedotorg ) and the Plant MicroRNA Database (PMRD, wwwdotbioinformaticsdotcaudotedudotcn/PMRD).
  • the mature miRNA sequences that are homologous or orthologous to the miRNAs of the invention (listed in Table 1) are found using miRNA public databases, having at least 90% identity of the entire small RNA length, and are summarized in Table 3 below.

Abstract

L'invention concerne des polynucléotides isolés exprimant ou modulant des microARNs ou des cibles de ceux-ci. Elle concerne également des plantes transgéniques les comprenant et des utilisations de ceux-ci pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante.
PCT/IB2011/054763 2010-10-25 2011-10-25 Polynucléotides isolés exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgéniques les comprenant et leurs utilisations pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante. WO2012056401A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US13/881,437 US20140013469A1 (en) 2010-10-25 2011-10-25 ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING microRNAs OR TARGETS OF SAME, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT
CA2815769A CA2815769A1 (fr) 2010-10-25 2011-10-25 Polynucleotides isoles exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgeniques les comprenant et leurs utilisations pour augmenter l'efficacite d'utilisation de l'azote, la tolerance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante
AU2011322146A AU2011322146A1 (en) 2010-10-25 2011-10-25 Isolated polynucleotides expressing or modulating microRNAs or targets of same, transgenic plants comprising same and uses thereof in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant
EP11799820.3A EP2633056A1 (fr) 2010-10-25 2011-10-25 Polynucléotides isolés exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgéniques les comprenant et leurs utilisations pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante
IL225964A IL225964A0 (en) 2010-10-25 2013-04-25 Isolated polynucleotides expressing or controlling microRNAs or their target proteins, transgenic plants containing them and their uses for improving nitrogen utilization, resistance to abiotic stresses, improving biomass, vigor and plant production

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US40618410P 2010-10-25 2010-10-25
US61/406,184 2010-10-25

Publications (1)

Publication Number Publication Date
WO2012056401A1 true WO2012056401A1 (fr) 2012-05-03

Family

ID=45401104

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2011/054763 WO2012056401A1 (fr) 2010-10-25 2011-10-25 Polynucléotides isolés exprimant ou modulant des microarns ou des cibles de ceux-ci, plantes transgéniques les comprenant et leurs utilisations pour augmenter l'efficacité d'utilisation de l'azote, la tolérance aux stress abiotiques, la biomasse, la vigueur ou le rendement d'une plante.

Country Status (6)

Country Link
US (1) US20140013469A1 (fr)
EP (1) EP2633056A1 (fr)
AU (1) AU2011322146A1 (fr)
CA (1) CA2815769A1 (fr)
IL (1) IL225964A0 (fr)
WO (1) WO2012056401A1 (fr)

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013175480A1 (fr) 2012-05-24 2013-11-28 A.B. Seeds Ltd. Compositions et procédés pour le silençage de l'expression génique
WO2014106837A2 (fr) 2013-01-01 2014-07-10 A. B. Seeds Ltd. Molécules d'arnds isolées et procédés pour les utiliser pour le silençage de molécules d'intérêt cibles
WO2014138339A2 (fr) 2013-03-07 2014-09-12 Athenix Corp. Gènes de toxine et leurs procédés d'utilisation
US9121022B2 (en) 2010-03-08 2015-09-01 Monsanto Technology Llc Method for controlling herbicide-resistant plants
US9416363B2 (en) 2011-09-13 2016-08-16 Monsanto Technology Llc Methods and compositions for weed control
US9422557B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9422558B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9540642B2 (en) 2013-11-04 2017-01-10 The United States Of America, As Represented By The Secretary Of Agriculture Compositions and methods for controlling arthropod parasite and pest infestations
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US9840715B1 (en) 2011-09-13 2017-12-12 Monsanto Technology Llc Methods and compositions for delaying senescence and improving disease tolerance and yield in plants
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US9920326B1 (en) 2011-09-14 2018-03-20 Monsanto Technology Llc Methods and compositions for increasing invertase activity in plants
USD818587S1 (en) 2016-03-29 2018-05-22 Abbevie Inc. Automatic injection device
US9981087B2 (en) 2011-09-22 2018-05-29 Abbvie Inc. Automatic injection device
WO2018098214A1 (fr) 2016-11-23 2018-05-31 Bayer Cropscience Lp Gènes de toxines axmi669 et axmi991 et procédés d'utilisation de ceux-ci
US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
WO2018119336A1 (fr) 2016-12-22 2018-06-28 Athenix Corp. Utilisation de cry14 dans la lutte contre les nématodes parasites
WO2018136611A1 (fr) 2017-01-18 2018-07-26 Bayer Cropscience Lp Utilisation de bp005 pour lutter contre des pathogènes végétaux
WO2018136604A1 (fr) 2017-01-18 2018-07-26 Bayer Cropscience Lp Gène de toxine bp005 et ses procédés d'utilisation
US10077451B2 (en) 2012-10-18 2018-09-18 Monsanto Technology Llc Methods and compositions for plant pest control
USD832424S1 (en) 2011-09-22 2018-10-30 Abbvie Inc. Automatic injection device
US10334848B2 (en) 2014-01-15 2019-07-02 Monsanto Technology Llc Methods and compositions for weed control using EPSPS polynucleotides
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10435701B2 (en) 2013-03-14 2019-10-08 Monsanto Technology Llc Methods and compositions for plant pest control
US10557138B2 (en) 2013-12-10 2020-02-11 Beeologics, Inc. Compositions and methods for virus control in Varroa mite and bees
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US10612019B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10609930B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10655136B2 (en) 2015-06-03 2020-05-19 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
CN111593058A (zh) * 2020-05-25 2020-08-28 扬州大学 Bna-miR169n基因及其在控制甘蓝型油菜抗旱性中的应用
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US10801028B2 (en) 2009-10-14 2020-10-13 Beeologics Inc. Compositions for controlling Varroa mites in bees
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10808249B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
US10883103B2 (en) 2015-06-02 2021-01-05 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
US10888579B2 (en) 2007-11-07 2021-01-12 Beeologics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
US10968449B2 (en) 2015-01-22 2021-04-06 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US10988764B2 (en) 2014-06-23 2021-04-27 Monsanto Technology Llc Compositions and methods for regulating gene expression via RNA interference
US11091770B2 (en) 2014-04-01 2021-08-17 Monsanto Technology Llc Compositions and methods for controlling insect pests
US11807857B2 (en) 2014-06-25 2023-11-07 Monsanto Technology Llc Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113215265B (zh) * 2021-03-25 2022-10-04 天津市农业科学院 奶牛bta-miRNA29d-3p在奶牛乳腺上皮细胞脂质积累调节过程中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133643A2 (fr) * 2006-10-12 2008-11-06 Monsanto Technology, Llc Microarn de plantes et leurs procédés d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008133643A2 (fr) * 2006-10-12 2008-11-06 Monsanto Technology, Llc Microarn de plantes et leurs procédés d'utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FRANCO-ZORRILLA JOSE MANUEL ET AL: "Target mimicry provides a new mechanism for regulation of microRNA activity", NATURE GENETICS, NATURE PUBLISHING GROUP, NEW YORK, US, vol. 39, no. 8, 1 August 2007 (2007-08-01), pages 1033 - 1037, XP002599594, ISSN: 1061-4036, [retrieved on 20070722], DOI: 10.1038/NG2079 *
OSWALDO VALDÉS-LÓPEZ ET AL: "MicroRNA expression profile in common bean (Phaseolus vulgaris) under nutrient deficiency stresses and manganese toxicity", NEW PHYTOLOGIST, vol. 187, no. 3, 1 August 2010 (2010-08-01), pages 805 - 818, XP055019664, ISSN: 0028-646X, DOI: 10.1111/j.1469-8137.2010.03320.x *
SHUKLA L I ET AL: "The role of microRNAs and other endogenous small RNAs in plant stress responses", BBA - GENE REGULATORY MECHANISMS, ELSEVIER, AMSTERDAM, NL, vol. 1779, no. 11, 1 November 2008 (2008-11-01), pages 743 - 748, XP025626372, ISSN: 1874-9399, [retrieved on 20080416], DOI: 10.1016/J.BBAGRM.2008.04.004 *
SUNKAR ET AL: "Small RNAs as big players in plant abiotic stress responses and nutrient deprivation", TRENDS IN PLANT SCIENCE, ELSEVIER SCIENCE, OXFORD, GB, vol. 12, no. 7, 1 July 2007 (2007-07-01), pages 301 - 309, XP022148764, ISSN: 1360-1385, DOI: 10.1016/J.TPLANTS.2007.05.001 *

Cited By (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10888579B2 (en) 2007-11-07 2021-01-12 Beeologics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
US10801028B2 (en) 2009-10-14 2020-10-13 Beeologics Inc. Compositions for controlling Varroa mites in bees
US11812738B2 (en) 2010-03-08 2023-11-14 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
US9988634B2 (en) 2010-03-08 2018-06-05 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
US9121022B2 (en) 2010-03-08 2015-09-01 Monsanto Technology Llc Method for controlling herbicide-resistant plants
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
US10808249B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US9422558B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9416363B2 (en) 2011-09-13 2016-08-16 Monsanto Technology Llc Methods and compositions for weed control
US10435702B2 (en) 2011-09-13 2019-10-08 Monsanto Technology Llc Methods and compositions for delaying senescence and improving disease tolerance and yield in plants
US9840715B1 (en) 2011-09-13 2017-12-12 Monsanto Technology Llc Methods and compositions for delaying senescence and improving disease tolerance and yield in plants
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US9422557B2 (en) 2011-09-13 2016-08-23 Monsanto Technology Llc Methods and compositions for weed control
US9920326B1 (en) 2011-09-14 2018-03-20 Monsanto Technology Llc Methods and compositions for increasing invertase activity in plants
US10428338B2 (en) 2011-09-14 2019-10-01 Monsanto Technology Llc Methods and compositions for increasing invertase activity in plants
USD832424S1 (en) 2011-09-22 2018-10-30 Abbvie Inc. Automatic injection device
US9981087B2 (en) 2011-09-22 2018-05-29 Abbvie Inc. Automatic injection device
US10857302B2 (en) 2011-09-22 2020-12-08 Abbvie Inc. Automatic injection device
US10934555B2 (en) 2012-05-24 2021-03-02 Monsanto Technology Llc Compositions and methods for silencing gene expression
WO2013175480A1 (fr) 2012-05-24 2013-11-28 A.B. Seeds Ltd. Compositions et procédés pour le silençage de l'expression génique
US10240161B2 (en) 2012-05-24 2019-03-26 A.B. Seeds Ltd. Compositions and methods for silencing gene expression
US10240162B2 (en) 2012-05-24 2019-03-26 A.B. Seeds Ltd. Compositions and methods for silencing gene expression
US10844398B2 (en) 2012-10-18 2020-11-24 Monsanto Technology Llc Methods and compositions for plant pest control
US10077451B2 (en) 2012-10-18 2018-09-18 Monsanto Technology Llc Methods and compositions for plant pest control
US10041068B2 (en) 2013-01-01 2018-08-07 A. B. Seeds Ltd. Isolated dsRNA molecules and methods of using same for silencing target molecules of interest
WO2014106837A2 (fr) 2013-01-01 2014-07-10 A. B. Seeds Ltd. Molécules d'arnds isolées et procédés pour les utiliser pour le silençage de molécules d'intérêt cibles
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
WO2014106838A2 (fr) 2013-01-01 2014-07-10 A.B. Seeds Ltd. Procédés d'introduction d'arnds dans des graines de plante pour moduler l'expression des gènes
US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
WO2014138339A2 (fr) 2013-03-07 2014-09-12 Athenix Corp. Gènes de toxine et leurs procédés d'utilisation
EP3626828A2 (fr) 2013-03-07 2020-03-25 BASF Agricultural Solutions Seed US LLC Gènes de toxine et leurs procédés d'utilisation
US10609930B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10612019B2 (en) 2013-03-13 2020-04-07 Monsanto Technology Llc Methods and compositions for weed control
US10435701B2 (en) 2013-03-14 2019-10-08 Monsanto Technology Llc Methods and compositions for plant pest control
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US11377667B2 (en) 2013-07-19 2022-07-05 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US9777288B2 (en) 2013-07-19 2017-10-03 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
US9856495B2 (en) 2013-07-19 2018-01-02 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US10597676B2 (en) 2013-07-19 2020-03-24 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US10927374B2 (en) 2013-11-04 2021-02-23 Monsanto Technology Llc Compositions and methods for controlling arthropod parasite and pest infestations
US9540642B2 (en) 2013-11-04 2017-01-10 The United States Of America, As Represented By The Secretary Of Agriculture Compositions and methods for controlling arthropod parasite and pest infestations
US10100306B2 (en) 2013-11-04 2018-10-16 Monsanto Technology Llc Compositions and methods for controlling arthropod parasite and pest infestations
US10557138B2 (en) 2013-12-10 2020-02-11 Beeologics, Inc. Compositions and methods for virus control in Varroa mite and bees
US10334848B2 (en) 2014-01-15 2019-07-02 Monsanto Technology Llc Methods and compositions for weed control using EPSPS polynucleotides
US11091770B2 (en) 2014-04-01 2021-08-17 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10988764B2 (en) 2014-06-23 2021-04-27 Monsanto Technology Llc Compositions and methods for regulating gene expression via RNA interference
US11807857B2 (en) 2014-06-25 2023-11-07 Monsanto Technology Llc Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
US11124792B2 (en) 2014-07-29 2021-09-21 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10378012B2 (en) 2014-07-29 2019-08-13 Monsanto Technology Llc Compositions and methods for controlling insect pests
US10968449B2 (en) 2015-01-22 2021-04-06 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
US10883103B2 (en) 2015-06-02 2021-01-05 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
US10655136B2 (en) 2015-06-03 2020-05-19 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
USD818587S1 (en) 2016-03-29 2018-05-22 Abbevie Inc. Automatic injection device
WO2018098214A1 (fr) 2016-11-23 2018-05-31 Bayer Cropscience Lp Gènes de toxines axmi669 et axmi991 et procédés d'utilisation de ceux-ci
WO2018119336A1 (fr) 2016-12-22 2018-06-28 Athenix Corp. Utilisation de cry14 dans la lutte contre les nématodes parasites
WO2018136611A1 (fr) 2017-01-18 2018-07-26 Bayer Cropscience Lp Utilisation de bp005 pour lutter contre des pathogènes végétaux
WO2018136604A1 (fr) 2017-01-18 2018-07-26 Bayer Cropscience Lp Gène de toxine bp005 et ses procédés d'utilisation
CN111593058A (zh) * 2020-05-25 2020-08-28 扬州大学 Bna-miR169n基因及其在控制甘蓝型油菜抗旱性中的应用

Also Published As

Publication number Publication date
AU2011322146A1 (en) 2013-06-06
CA2815769A1 (fr) 2012-05-03
IL225964A0 (en) 2013-06-27
US20140013469A1 (en) 2014-01-09
EP2633056A1 (fr) 2013-09-04

Similar Documents

Publication Publication Date Title
US11453887B2 (en) Isolated polypeptides and polynucleotides useful for increasing nitrogen use efficiency, abiotic stress tolerance, yield and biomass in plants
US20210115460A1 (en) Isolated polynucleotides and polypeptides and methods of using same for increasing plant utility
US20190085354A1 (en) Isolated polynucleotides expressing or modulating micrornas or targets of same, transgenic plants comprising same and uses thereof
US20140013469A1 (en) ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING microRNAs OR TARGETS OF SAME, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT
US9562235B2 (en) MicroRNA compositions and methods for enhancing plant resistance to abiotic stress
US9902956B2 (en) Nucleic acid agents for overexpressing or downregulating RNA interference targets and uses of same in improving nitrogen use efficiency, abiotic stress tolerance, biomass, vigor or yield of a plant
US20140317781A1 (en) Isolated polynucleotides and polypeptides, transgenic plants comprising same and uses thereof in improving abiotic stress tolerance, nitrogen use efficiency, biomass, vigor or yield of plants
US20170218388A1 (en) Method of improving abiotic stress tolerance of plants and plants generated thereby
US20140298541A1 (en) ISOLATED POLYNUCLEOTIDES EXPRESSING OR MODULATING dsRNAs, TRANSGENIC PLANTS COMPRISING SAME AND USES THEREOF IN IMPROVING NITROGEN USE EFFICIENCY, ABIOTIC STRESS TOLERANCE, BIOMASS, VIGOR OR YIELD OF A PLANT
AU2018200117B2 (en) Isolated Polynucleotides and Polypeptides and Methods of Using Same for Increasing Plant Utility

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11799820

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2815769

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 13881437

Country of ref document: US

Ref document number: 225964

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 2011799820

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2011322146

Country of ref document: AU

Date of ref document: 20111025

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112013010051

Country of ref document: BR

ENPW Started to enter national phase and was withdrawn or failed for other reasons

Ref document number: 112013010051

Country of ref document: BR

Free format text: TENDO EM VISTA MANIFESTACAO EXPRESSA DO REQUERENTE, EM RESPOSTA AO COMUNICADO PUBLICADO NA RPI 2612, DE 26/01/2021, INFORMANDO O ABANDONO/DESISTENCIA DO PEDIDO, PUBLICA-SE A RETIRADA DO PEDIDO (1.2).

ENPZ Former announcement of the withdrawal of the entry into the national phase was wrong

Ref document number: 112013010051

Country of ref document: BR

Free format text: ANULADA A PUBLICACAO CODIGO 1.2 NA RPI NO 2635 DE 06/07/2021 POR TER SIDO INDEVIDA.